This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Study of the lipid targets of CD1c-mediated T cell responses recently identified a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponded to mammalian mannosyl b-1-phosphodolichols (MPD), but contained an unusual lipid moiety. We have shown that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C30-34) and are produced by M. tuberculosis, M. bovis Bacille-Calmette-Guerin and other mycobacteria that grow within cells. Analysis of the fine structures of these mycobacterial antigens showed that the chemical features that distinguished them from mammalian MPDs were necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway. Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids in C5 increments by strictly alternating incorporation of C2 and C3 units, rather than isopentenyl pyrophosphate condensation. Inspection of the M. tuberculosis genome identified one gene pks12, which is predicted to make the largest protein in the proteome and to contain 12 catalytic domains necessary to carry out this multi-step synthesis. Genetic deletion and complementation showed that PKS12 was necessary and sufficient for antigen production and the resulting CD1c-mediated T cell activation, but did not affect synthesis of true isoprenols. These studies establish the genetic and enzymatic basis for a previously unknown type of mycobacterial polyketide, designated mycoketide, which represents a lipidic pathogen associated molecular pattern that allows mycobacterial recognition by the human immune system. These compounds provide new iinsight into the potential mechanism of CD1c-restricted T cell activation;they may bear relevance to prevention or treatment of AIDS. A series of synthetic compounds has been prepared and the manuscript describing their properties has been accepted for publication.